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Cell fate control gene therapy based on engineered variants of human deoxycytidine kinase.

ABSTRACT: The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.

SUBMITTER: Neschadim A

PROVIDER: S-EPMC3345984 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structural and kinetic characterization of human deoxycytidine kinase variants able to phosphorylate 5-substituted deoxycytidine and thymidine analogues .

Project description:The physiological role of human deoxycytidine kinase (dCK) is to phosphorylate deoxynucleosides required for DNA synthesis, with the exception of thymidine. Previous structural analysis of dCK implicated steric factors, specifically the thymine methyl group at the 5-position, that prevent thymidine phosphorylation by dCK. This hypothesis is supported by the observation that mutations that enlarge the active site cavity in proximity to the nucleoside 5-position endow dCK with the ability to phosphorylate thymidine. However, in conflict with this hypothesis was our discovery that the cytidine analogue 5-methyldeoxycytidine (5-Me-dC), an isostere of thymidine, can indeed be phosphorylated by wild-type (WT) dCK. To reconcile this seemingly contradicting observation, and to better understand the determinants preventing thymidine phosphorylation by WT dCK, we solved the crystal structure of dCK in complex with 5-Me-dC. The structure reveals the active site adjustments required to accommodate the methyl group at the 5-position. Combination of kinetic, mutagenesis, and structural data suggested that it is in fact residue Asp133 of dCK that is most responsible for discriminating against the thymine base. dCK variants in which Asp133 is replaced by an alanine and Arg104 by select hydrophobic residues attain significantly improved activity with 5-substituted deoxycytidine and thymidine analogues. Importantly, the ability of the designer enzymes to activate 5-substitued pyrimidines makes it possible to utilize such nucleoside analogues in suicide gene therapy or protein therapy applications that target cancer cells.

| S-EPMC2925221 | biostudies-literature

Genomic structure and chromosomal localization of the human deoxycytidine kinase gene.

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| S-EPMC45676 | biostudies-other

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A new pyrimidine-specific reporter gene: a mutated human deoxycytidine kinase suitable for PET during treatment with acycloguanosine-based cytotoxic drugs.

Project description:In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET.Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs.Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine).The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

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Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy.

Project description:The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their "stem-like" properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.

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Cell Fate Control by Engineered Chromatin Regulator

Project description:MS analysis to provide insights into proteins bound to engineered cell fate regulator NanogBiD during somatic cell reprogramming

2023-10-09 | PXD046008 |

Cloning and expression of human deoxycytidine kinase cDNA.

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| S-EPMC6522309 | biostudies-literature

Deoxycytidine production by a metabolically engineered Escherichia coli strain.

Project description:Rational engineering studies for deoxycytidine production were initiated due to low intracellular levels and tight regulation. To achieve high-level production of deoxycytidine, a useful precursor of decitabine, genes related to feed-back inhibition as well as the biosynthetic pathway were engineered. Additionally, we predicted the impact of individual gene expression levels on a complex metabolic network by microarray analysis. Based on these findings, we demonstrated rational metabolic engineering strategies capable of producing deoxycytidine.To prepare the deoxycytidine producing strain, we first deleted 3 degradation enzymes in the salvage pathway (deoA, udp, and deoD) and 4 enzymes involved in the branching pathway (dcd, cdd, codA and thyA) to completely eliminate degradation of deoxycytidine. Second, purR, pepA and argR were knocked out to prevent feedback inhibition of CarAB. Third, to enhance influx to deoxycytidine, we investigated combinatorial expression of pyrG, T4 nrdCAB and yfbR. The best strain carried pETGY (pyrG-yfbR) from the possible combinatorial plasmids. The resulting strain showed high deoxycytidine yield (650 mg/L) but co-produced byproducts. To further improve deoxycytidine yield and reduce byproduct formation, pgi was disrupted to generate a sufficient supply of NADPH and ribose. Overall, in shake-flask cultures, the resulting strain produced 967 mg/L of dCyd with decreased byproducts.We demonstrated that deoxycytidine could be readily achieved by recombineering with biosynthetic genes and regulatory genes, which appeared to enhance the supply of precursors for synthesis of carbamoyl phosphate, based on transcriptome analysis. In addition, we showed that carbon flux rerouting, by disrupting pgi, efficiently improved deoxycytidine yield and decreased byproduct content.

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